1. Field of the Invention
The present invention relates to methods for identifying modulators of a regulator of G-protein signaling (RGS) protein. The present invention also relates to compositions comprising an RGS modulatory compound, and to methods of using such compositions to modulate G protein-coupled receptor (GPCR) signal transduction. In particular, the present invention relates to methods for identifying compounds that specifically inhibit or enhance the activity of an RGS21 protein or biologically active fragment thereof, compositions comprising such a compound; and methods of using such a compound to modulate taste signal transduction through GPCR taste signal transduction processes.
2. Background
G protein-coupled receptors (GPCRs) play a major role in signal transduction and are targets of many therapeutic drugs. It has been reported for a long time that the standard model of GPCR signal transduction is restricted to a three-component system: G-protein coupled receptor (GPCR), G protein, and effector (Neubig and Siderovski, 2002, Nat. Rev. Drug Discov. 1(3):187-97). GPCRs are cell-surface receptor proteins having seven transmembrane domains. Each G protein is a membrane-associated heterotrimeric complex that comprises a GTP-hydrolysing Gα subunit and a Gβγ dimeric subunit. Gα subunits are molecular switches that control a broad range of physiological processes in cells. Gα subunits exist in an inactive, GDP-bound state or in an activated, GTP-bound state where they interact with downstream signaling proteins to elicit a specific signaling response (FIG. 1). The activation and inactivation of Gα proteins are regulated by ligand-bound GPCRs and GTPase accelerating proteins (GAPs), respectively. GPCRs promote cell signaling, upon binding to a ligand, by catalyzing the exchange of guanosine tri-phosphate (GTP) for guanosine di-phosphate (GDP) onto the α subunit of heterotrimeric G proteins. GAPs bind to the active, GTP-bound form of the Gα protein and stimulate the G protein's intrinsic GTPase activity, whereby the terminal phosphate residue of the bound GTP is hydrolyzed to GDP, thus returning the Gα protein to the inactive state (FIG. 1).
When an agonist binds to a GPCR, it causes conformational changes that enhance the guanine-nucleotide-exchange activity of the GPCR, leading to the release of GDP (and subsequent binding to GTP) by the Gα subunit. On binding to GTP, conformational changes within the three ‘switch’ regions of the Cα subunit allow the release of the Gβγ subunits and the subsequent engagement of effectors that are specific to each Gα subtype. Freed Gβγ subunits also can modulate effectors, including ion channels and specific isoforms of adenylyl cyclase and phospholipase (PLC) (Neubig and Siderovski, 2002, Nat. Rev. Drug Discov. 1(3):187-97, FIG. 1 and Table 1).
Recently, a protein family has been discovered that acts as a new component of GPCR signal transduction. This protein family consists of proteins known as regulator of G protein signaling (RGS) proteins (DeVries et al., 2000, Ann. Rev. Pharmacol. 40:235; Ross and Wilkie, 2001, Ann. Rev. Biochem. 69:795). RGS proteins strongly modulate the activity of G proteins and play a key role in GPCR signal transduction. Their best-known function is to act as a GTPase activating protein (GAP), inhibiting G protein signaling by accelerating GTP hydrolysis, and thus turning off G protein signals. In particular, RGS proteins control the output of signaling by an activated Gα subunit by directly binding to the GTP-bound Gα subunit. This binding markedly accelerates the subunit's rate of GTP hydrolysis, and therefore, the rate of inactivation of GPCR signaling (Neubig and Siderovski, 2002, Nat. Rev. Drug Discov. 1(3):187-97; Berman et al., 1996, Cell 86: 445-52; Hunt et al., 1996, Nature 383:175-77; Watson et al., 1996, Nature 383:172-75).
There are at least 37 RGS proteins present in the human genome, and these can be subdivided into distinct protein families which differ in the composition of their functional domains (FIG. 2). All RGS proteins contain at least one conserved domain of approximately 120 amino acids called the “RGS-box,” which is responsible for the observed GAP activity of RGS proteins (FIG. 3). The RGS-box contacts the Gα switch regions to stabilize their configuration in the transition state between GTP-bound and GDP-bound forms. Because RGS proteins are highly diverse, have unique tissue distributions, and play diverse functional roles in living cells, RGS proteins typically also contain various non-RGS-box domains and motifs (e.g., GGL, DEP, DR/PH, PDZ domains, and a cysteine string motif).
RGS proteins negatively regulate GPCR signaling, and therefore, RGS proteins have been considered to be potential drug discovery targets because the inhibition of RGS-box GAP activity should lead to prolonged and enhanced signaling from agonist-bound GPCRs Neubig and Siderovski, 2002, Nat. Rev. Drug Discov. 1(3):187-97). Inhibitors of RGS proteins may enhance G protein signaling by impairing the inactivation of Gα protein. The potential therapeutic roles of RGS inhibitors include, but are not limited to, enhanced unction of endogenous neurotransmitters; enhanced function of exogenous GPCR-agonist drugs; reduced desensitization to agonist drugs; modified specificity of exogenous agonists; and blocked regulation of RGS-protein-mediated effector activity Neubig and Siderovski, 2002, Nat. Rev. Drug Discov. 1(3):187-97, Box 1; Zhong & Neubig, 2001, J. Pharmacol. Exp. Ther. 297:837-45).
Several RGS genes have been found in the central nervous system (CNS), providing potential drug targets for the clinical use of RGS inhibitors for CNS diseases, such as Alzheimer's disease, depression, epilepsy, Parkinson's disease, pain, and spasticity Neubig and Siderovski, 2002, Nat. Rev. Drug Discov. 1(3):187-97, Tables 3 and 4). However, because of the high diversity and complexity of RGS proteins, the effects of each RGS protein may depend on the function of the particular domains, including the RGS-box, non-RGS-box motifs, and/or other functional modules ((Neubig and Siderovski, 2002, Nat. Rev. Drug Discov. 1(3):187-97).
Taste cells are assembled into taste buds on the tongue surface (Lindemann, 1996, Physiol. Rev. 76:718-66). Two families of GPCRs have been identified in taste cells: the T1R family of GPCRs that mediates sweet and umami tastes, and the T2R family of GPCRs that mediate bitter tastes (Nelson et al., 2001, Cell 106:381-90; Nelson et al., 2002, Nature 416:199-202; Li et al., 2002, Proc. Natl. Acad. Sci. USA 99:4692-96; Zhao et al., 2003, Cell 115:255-66; Adler et al., 2000, Cell 100: 693-702; Chandrashekar et al., 2000, Cell 100:703-11; Bufe et al., 2002, Nat. Genet. 32:397-401). Signaling downstream of all of these receptors has been shown to depend on the key effector enzyme of sweet, umami, and bitter taste transduction, phospholipase C subtype β2 (PLCβ2), and the trp channel subtype m5 (TRPM5) (Zhang et al., 2003, Cell 112:293-301).
Buccholtz et al. identified another ROS protein, RGS21, and demonstrated that RGS21 is specifically expressed in foliate, fungiform, and circumvallate taste bud cells, where it co-localizes with bitter receptors (T2R), umami receptors (T1R1/T1R3), sweetener receptors (T1R2/T1R3), α-gustducin, and phospholipase Cβ2 (PLCβ2). Buchholtz et al. also showed that RGS21 protein can associate with Gαi/o/t/z, Gq/11/14, and α-gustducin. Sequence analysis of human RGS21 indicates that it contains a single RGS-box domain and no other functional domains. Furthermore, the sequence homology of the RGS-box of RGS21 to that of RGS2, a GAP for Gi/o, and Gq proteins, further supports the possibility that RGS21 similarly regulates these G-proteins (FIG. 3). By analogy with other RGS proteins, it is likely possible, although not yet demonstrated, that RGS21 protein attenuates α-gustducin and/or other relevant Gα proteins that participate in taste cell signaling.
What is needed in the art are methods for identifying compounds that are useful for modulating taste signal transduction. Also needed are compounds that modulate taste signal transduction and methods of using such compounds for the modulation of taste signal transduction.